Spatial transcriptomic data with the Visium platform is in many ways similar to scRNAseq data. It contains UMI counts for 5-20 cells instead of single cells, but is still quite sparse in the same way as scRNAseq data is, but with the additional information about spatial location in the tissue.
Here we will first run quality control in a similar manner to scRNAseq data, then QC filtering, dimensionality reduction, integration and clustering. Then we will use scRNAseq data from mouse cortex to run LabelTransfer to predict celltypes in the Visium spots.
We will use two Visium spatial transcriptomics dataset of the mouse brain (Sagittal), which are publicly available from the 10x genomics website. Note, that these dataset have already been filtered for spots that does not overlap with the tissue.
mkdir -p data/visium/Posterior
mkdir -p data/visium/Anterior
cd data/visium/Posterior
curl -o V1_Mouse_Brain_Sagittal_Posterior_filtered_feature_bc_matrix.tar.gz https://cf.10xgenomics.com/samples/spatial-exp/1.1.0/V1_Mouse_Brain_Sagittal_Posterior/V1_Mouse_Brain_Sagittal_Posterior_filtered_feature_bc_matrix.tar.gz
tar xvzf V1_Mouse_Brain_Sagittal_Posterior_filtered_feature_bc_matrix.tar.gz
curl -o V1_Mouse_Brain_Sagittal_Posterior_spatial.tar.gz https://cf.10xgenomics.com/samples/spatial-exp/1.1.0/V1_Mouse_Brain_Sagittal_Posterior/V1_Mouse_Brain_Sagittal_Posterior_spatial.tar.gz
tar xvzf V1_Mouse_Brain_Sagittal_Posterior_spatial.tar.gz
rm *.tar.gz
cd ../Anterior
curl -o V1_Mouse_Brain_Sagittal_Anterior_filtered_feature_bc_matrix.tar.gz https://cf.10xgenomics.com/samples/spatial-exp/1.1.0/V1_Mouse_Brain_Sagittal_Anterior/V1_Mouse_Brain_Sagittal_Anterior_filtered_feature_bc_matrix.tar.gz
tar xvzf V1_Mouse_Brain_Sagittal_Anterior_filtered_feature_bc_matrix.tar.gz
curl -o V1_Mouse_Brain_Sagittal_Anterior_spatial.tar.gz https://cf.10xgenomics.com/samples/spatial-exp/1.1.0/V1_Mouse_Brain_Sagittal_Anterior/V1_Mouse_Brain_Sagittal_Anterior_spatial.tar.gz
tar xvzf V1_Mouse_Brain_Sagittal_Anterior_spatial.tar.gz
rm *.tar.gz
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## x filtered_feature_bc_matrix/
## x filtered_feature_bc_matrix/barcodes.tsv.gz
## x filtered_feature_bc_matrix/matrix.mtx.gz
## x filtered_feature_bc_matrix/features.tsv.gz
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## x spatial/
## x spatial/tissue_positions_list.csv
## x spatial/tissue_hires_image.png
## x spatial/scalefactors_json.json
## x spatial/aligned_fiducials.jpg
## x spatial/detected_tissue_image.jpg
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## x filtered_feature_bc_matrix/
## x filtered_feature_bc_matrix/barcodes.tsv.gz
## x filtered_feature_bc_matrix/features.tsv.gz
## x filtered_feature_bc_matrix/matrix.mtx.gz
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## x spatial/
## x spatial/tissue_positions_list.csv
## x spatial/tissue_hires_image.png
## x spatial/scalefactors_json.json
## x spatial/detected_tissue_image.jpg
## x spatial/tissue_lowres_image.png
## x spatial/aligned_fiducials.jpg
devtools::install_github("RachelQueen1/Spaniel", ref = "Development")
library(Spaniel)
library(biomaRt)
suppressPackageStartupMessages(require(Matrix))
suppressPackageStartupMessages(require(dplyr))
suppressPackageStartupMessages(require(scran))
suppressPackageStartupMessages(require(SingleR))
suppressPackageStartupMessages(require(scater))
suppressPackageStartupMessages(require(ggplot2))
suppressPackageStartupMessages(require(patchwork))
suppressPackageStartupMessages(require(cowplot))We can first load and merge the objects into one SCE object.
sce.a <- Spaniel::createVisiumSCE(tenXDir = "data/visium/Anterior", resolution = "Low")
sce.p <- Spaniel::createVisiumSCE(tenXDir = "data/visium/Posterior", resolution = "Low")
sce <- cbind(sce.a, sce.p)
sce$Sample <- sub(".*[/]", "", sub("/filtered_feature_bc_matrix", "", sce$Sample))
lll <- list(sce.a, sce.p)
lll <- lapply(lll, function(x) x@metadata)
names(lll) <- c("Anterior", "Posterior")
sce@metadata <- lllWe can further convert the gene ensembl IDs to gene names.
mart <- biomaRt::useMart(biomart = "ENSEMBL_MART_ENSEMBL", dataset = "mmusculus_gene_ensembl",
host = "https://nov2020.archive.ensembl.org")
annot <- biomaRt::getBM(attributes = c("ensembl_gene_id", "external_gene_name", "gene_biotype"),
mart = mart)
gene_names <- as.character(annot[match(rownames(sce), annot[, "ensembl_gene_id"]),
"external_gene_name"])
gene_names[is.na(gene_names)] <- ""
sce <- sce[gene_names != "", ]
rownames(sce) <- gene_names[gene_names != ""]
dim(sce)## [1] 32275 6050
##Quality control ***
Similar to scRNAseq we use statistics on number of counts, number of features and percent mitochondria for quality control.
Now the counts and feature counts are calculated on the Spatial assay, so they are named “nCount_Spatial” and “nFeature_Spatial”.
# Mitochondrial genes
mito_genes <- rownames(sce)[grep("^mt-", rownames(sce))]
# Ribosomal genes
ribo_genes <- rownames(sce)[grep("^Rp[sl]", rownames(sce))]
# Hemoglobin genes - includes all genes starting with HB except HBP.
hb_genes <- rownames(sce)[grep("^Hb[^(p)]", rownames(sce))]
sce <- addPerCellQC(sce, flatten = T, subsets = list(mt = mito_genes, hb = hb_genes,
ribo = ribo_genes))
head(colData(sce))## DataFrame with 6 rows and 24 columns
## Sample Barcode Section Spot_Y Spot_X Image_Y
## <character> <character> <integer> <integer> <integer> <integer>
## 1 Anterior AAACAAGTATCTCCCA-1 1 50 102 7474
## 2 Anterior AAACACCAATAACTGC-1 1 59 19 8552
## 3 Anterior AAACAGAGCGACTCCT-1 1 14 94 3163
## 4 Anterior AAACAGCTTTCAGAAG-1 1 43 9 6636
## 5 Anterior AAACAGGGTCTATATT-1 1 47 13 7115
## 6 Anterior AAACATGGTGAGAGGA-1 1 62 0 8912
## Image_X pixel_x pixel_y sum detected total sum
## <integer> <numeric> <numeric> <numeric> <integer> <numeric> <numeric>
## 1 8500 438.898 214.079 13991 4462 13991 13963
## 2 2788 143.959 158.417 39797 8126 39797 39764
## 3 7950 410.499 436.678 29951 6526 29951 29919
## 4 2100 108.434 257.349 42333 8190 42333 42293
## 5 2375 122.633 232.616 35700 8090 35700 35686
## 6 1480 76.420 139.828 22148 6518 22148 22115
## detected subsets_mt_sum subsets_mt_detected subsets_mt_percent
## <integer> <numeric> <integer> <numeric>
## 1 4461 1521 12 10.89307
## 2 8124 3977 12 10.00151
## 3 6525 4265 12 14.25516
## 4 8187 2870 12 6.78599
## 5 8089 1831 13 5.13086
## 6 6517 2390 12 10.80714
## subsets_hb_sum subsets_hb_detected subsets_hb_percent subsets_ribo_sum
## <numeric> <integer> <numeric> <numeric>
## 1 60 4 0.429707 826
## 2 831 6 2.089830 2199
## 3 111 5 0.371002 1663
## 4 117 5 0.276642 3129
## 5 73 5 0.204562 2653
## 6 134 5 0.605924 1478
## subsets_ribo_detected subsets_ribo_percent total
## <integer> <numeric> <numeric>
## 1 85 5.91563 13963
## 2 89 5.53013 39764
## 3 88 5.55834 29919
## 4 88 7.39839 42293
## 5 90 7.43429 35686
## 6 84 6.68325 22115
plot_grid(plotColData(sce, y = "detected", x = "Sample", colour_by = "Sample"), plotColData(sce,
y = "total", x = "Sample", colour_by = "Sample"), plotColData(sce, y = "subsets_mt_percent",
x = "Sample", colour_by = "Sample"), plotColData(sce, y = "subsets_ribo_percent",
x = "Sample", colour_by = "Sample"), plotColData(sce, y = "subsets_hb_percent",
x = "Sample", colour_by = "Sample"), ncol = 3)We can also plot the same data onto the tissue section.
samples <- c("Anterior", "Posterior")
to_plot <- c("detected", "total", "subsets_mt_percent", "subsets_ribo_percent", "subsets_hb_percent")
plist <- list()
n = 1
for (j in to_plot) {
for (i in samples) {
temp <- sce[, sce$Sample == i]
temp@metadata <- temp@metadata[[i]]
plist[[n]] <- spanielPlot(object = temp, plotType = "Cluster", clusterRes = j,
customTitle = j, techType = "Visium", ptSizeMax = 1, ptSizeMin = 0.1)
n <- n + 1
}
}
plot_grid(ncol = 2, plotlist = plist)As you can see, the spots with low number of counts/features and high mitochondrial content is mainly towards the edges of the tissue. It is quite likely that these regions are damaged tissue. You may also see regions within a tissue with low quality if you have tears or folds in your section.
But remember, for some tissue types, the amount of genes expressed and proportion mitochondria may also be a biological features, so bear in mind what tissue you are working on and what these features mean.
Select all spots with less than 25% mitocondrial reads, less than 20% hb-reads and 1000 detected genes. You must judge for yourself based on your knowledge of the tissue what are appropriate filtering criteria for your dataset.
sce <- sce[, sce$detected > 500 & sce$subsets_mt_percent < 25 & sce$subsets_hb_percent <
20]
dim(sce)## [1] 32275 5805
And replot onto tissue section:
samples <- c("Anterior", "Posterior")
to_plot <- c("detected", "total", "subsets_mt_percent", "subsets_mt_percent", "subsets_hb_percent")
plist <- list()
n = 1
for (j in to_plot) {
for (i in samples) {
temp <- sce[, sce$Sample == i]
temp@metadata <- temp@metadata[[i]]
plist[[n]] <- spanielPlot(object = temp, plotType = "Cluster", clusterRes = j,
customTitle = j, techType = "Visium", ptSizeMax = 1, ptSizeMin = 0.1)
n <- n + 1
}
}
plot_grid(ncol = 2, plotlist = plist)As for scRNAseq data, we will look at what the top expressed genes are.
C = counts(sce)
C@x = C@x/rep.int(colSums(C), diff(C@p))
most_expressed <- order(Matrix::rowSums(C), decreasing = T)[20:1]
boxplot(as.matrix(t(C[most_expressed, ])), cex = 0.1, las = 1, xlab = "% total count per cell",
col = (scales::hue_pal())(20)[20:1], horizontal = TRUE)rm(C)As you can see, the mitochondrial genes are among the top expressed. Also the lncRNA gene Bc1 (brain cytoplasmic RNA 1). Also one hemoglobin gene.
We will remove the Bc1 gene, hemoglobin genes (blood contamination) and the mitochondrial genes.
dim(sce)## [1] 32275 5805
# Filter Bl1
sce <- sce[!grepl("Bc1", rownames(sce)), ]
# Filter Mitocondrial
sce <- sce[!grepl("^mt-", rownames(sce)), ]
# Filter Hemoglobin gene (optional if that is a problem on your data)
sce <- sce[!grepl("^Hb.*-", rownames(sce)), ]
dim(sce)## [1] 32253 5805
sce <- computeSumFactors(sce, sizes = c(20, 40, 60, 80))
sce <- logNormCounts(sce)Now we can plot gene expression of individual genes, the gene Hpca is a strong hippocampal marker and Ttr is a marker of the choroid plexus.
samples <- c("Anterior", "Posterior")
to_plot <- c("Hpca", "Ttr")
plist <- list()
n = 1
for (j in to_plot) {
for (i in samples) {
temp <- sce[, sce$Sample == i]
temp@metadata <- temp@metadata[[i]]
plist[[n]] <- spanielPlot(object = temp, plotType = "Gene", gene = j, customTitle = j,
techType = "Visium", ptSizeMax = 1, ptSizeMin = 0.1)
n <- n + 1
}
}
plot_grid(ncol = 2, plotlist = plist)We can then now run dimensionality reduction and clustering using the same workflow as we use for scRNA-seq analysis.
But make sure you run it on the SCT assay.
var.out <- modelGeneVar(sce, method = "loess")
hvgs = getTopHVGs(var.out, n = 2000)
sce <- runPCA(sce, exprs_values = "logcounts", subset_row = hvgs, ncomponents = 50,
ntop = 100, scale = T)
g <- buildSNNGraph(sce, k = 5, use.dimred = "PCA")
sce$louvain_SNNk5 <- factor(igraph::cluster_louvain(g)$membership)
sce <- runUMAP(sce, dimred = "PCA", n_dimred = 50, ncomponents = 2, min_dist = 0.1,
spread = 0.3, metric = "correlation", name = "UMAP_on_PCA")We can then plot clusters onto umap or onto the tissue section.
samples <- c("Anterior", "Posterior")
to_plot <- c("louvain_SNNk5")
plist <- list()
n = 1
for (j in to_plot) {
for (i in samples) {
temp <- sce[, sce$Sample == i]
temp@metadata <- temp@metadata[[i]]
plist[[n]] <- spanielPlot(object = temp, plotType = "Cluster", clusterRes = j,
customTitle = j, techType = "Visium", ptSizeMax = 1, ptSizeMin = 0.1)
n <- n + 1
}
}
plist[[3]] <- plotReducedDim(sce, dimred = "UMAP_on_PCA", colour_by = "louvain_SNNk5")
plist[[4]] <- plotReducedDim(sce, dimred = "UMAP_on_PCA", colour_by = "Sample")
plot_grid(ncol = 2, plotlist = plist)Quite often there are strong batch effects between different ST sections, so it may be a good idea to integrate the data across sections.
We will do a similar integration as in the Data Integration lab.
mnn_out <- batchelor::fastMNN(sce, subset.row = hvgs, batch = factor(sce$Sample),
k = 20, d = 50)
reducedDim(sce, "MNN") <- reducedDim(mnn_out, "corrected")
rm(mnn_out)
gc()## used (Mb) gc trigger (Mb) max used (Mb)
## Ncells 8678320 463.5 15881785 848.2 12399795 662.3
## Vcells 189347732 1444.7 332601989 2537.6 332601697 2537.6
Then we run dimensionality reduction and clustering as before.
g <- buildSNNGraph(sce, k = 5, use.dimred = "MNN")
sce$louvain_SNNk5 <- factor(igraph::cluster_louvain(g)$membership)
sce <- runUMAP(sce, dimred = "MNN", n_dimred = 50, ncomponents = 2, min_dist = 0.1,
spread = 0.3, metric = "correlation", name = "UMAP_on_MNN")samples <- c("Anterior", "Posterior")
to_plot <- c("louvain_SNNk5")
plist <- list()
n = 1
for (j in to_plot) {
for (i in samples) {
temp <- sce[, sce$Sample == i]
temp@metadata <- temp@metadata[[i]]
plist[[n]] <- spanielPlot(object = temp, plotType = "Cluster", clusterRes = j,
customTitle = j, techType = "Visium", ptSizeMax = 1, ptSizeMin = 0.1)
n <- n + 1
}
}
plist[[3]] <- plotReducedDim(sce, dimred = "UMAP_on_MNN", colour_by = "louvain_SNNk5")
plist[[4]] <- plotReducedDim(sce, dimred = "UMAP_on_MNN", colour_by = "Sample")
plot_grid(ncol = 2, plotlist = plist)Do you see any differences between the integrated and non-integrated clusering? Judge for yourself, which of the clusterings do you think looks best? As a reference, you can compare to brain regions in the Allen brain atlas.
There are two main workflows to identify molecular features that correlate with spatial location within a tissue. The first is to perform differential expression based on spatially distinct clusters, the other is to find features that are have spatial patterning without taking clusters or spatial annotation into account.
First, we will do differential expression between clusters just as we did for the scRNAseq data before.
# differential expression between cluster 4 and cluster 6
cell_selection <- sce[, sce$louvain_SNNk5 %in% c(4, 6)]
cell_selection$louvain_SNNk5 <- factor(cell_selection$louvain_SNNk5)
markers_genes <- scran::findMarkers(x = cell_selection, groups = cell_selection$louvain_SNNk5,
lfc = 0.25, pval.type = "all", direction = "up")
# List of dataFrames with the results for each cluster
top5_cell_selection <- lapply(names(markers_genes), function(x) {
temp <- markers_genes[[x]][1:5, 1:2]
temp$gene <- rownames(markers_genes[[x]])[1:5]
temp$cluster <- x
return(temp)
})
top5_cell_selection <- as_tibble(do.call(rbind, top5_cell_selection))
top5_cell_selection# plot top markers
samples <- c("Anterior", "Posterior")
to_plot <- top5_cell_selection$gene[1:5]
plist <- list()
n = 1
for (j in to_plot) {
for (i in samples) {
temp <- sce[, sce$Sample == i]
temp@metadata <- temp@metadata[[i]]
plist[[n]] <- spanielPlot(object = temp, plotType = "Gene", gene = j, customTitle = j,
techType = "Visium", ptSizeMax = 1, ptSizeMin = 0.1)
n <- n + 1
}
}
plot_grid(ncol = 2, plotlist = plist)We can also perform data integration between one scRNA-seq dataset and one spatial transcriptomics dataset. Such task is particularly useful because it allows us to transfer cell type labels to the Visium dataset, which were dentified from the scRNA-seq dataset.
We will use a reference scRNA-seq dataset of ~14,000 adult mouse cortical cell taxonomy from the Allen Institute, generated with the SMART-Seq2 protocol.
First dowload the seurat data from: https://www.dropbox.com/s/cuowvm4vrf65pvq/allen_cortex.rds?dl=1 to folder data/spatial/ with command:
FILE="./data/spatial/allen_cortex.rds"
if [ -e $FILE ]
then
echo "File $FILE is downloaded."
else
echo "Downloading $FILE"
mkdir -p data/spatial
wget -O data/spatial/allen_cortex.rds https://www.dropbox.com/s/cuowvm4vrf65pvq/allen_cortex.rds?dl=1
fi## File ./data/spatial/allen_cortex.rds is downloaded.
For speed, and for a more fair comparison of the celltypes, we will subsample all celltypes to a maximum of 200 cells per class (subclass).
allen_reference <- readRDS("data/spatial/allen_cortex.rds")
allen_reference_sce <- Seurat::as.SingleCellExperiment(allen_reference)
# check number of cells per subclass
allen_reference_sce$subclass <- sub("/", "_", sub(" ", "_", allen_reference_sce$subclass))
table(allen_reference_sce$subclass)##
## Astro CR Endo L2_3_IT L4 L5_IT L5_PT
## 368 7 94 982 1401 880 544
## L6_CT L6_IT L6b Lamp5 Macrophage Meis2 NP
## 960 1872 358 1122 51 45 362
## Oligo Peri Pvalb Serpinf1 SMC Sncg Sst
## 91 32 1337 27 55 125 1741
## Vip VLMC
## 1728 67
# select 20 cells per subclass, fist set subclass ass active.ident
subset_cells <- lapply(unique(allen_reference_sce$subclass), function(x) {
if (sum(allen_reference_sce$subclass == x) > 20) {
temp <- sample(colnames(allen_reference_sce)[allen_reference_sce$subclass ==
x], size = 20)
} else {
temp <- colnames(allen_reference_sce)[allen_reference_sce$subclass == x]
}
})
allen_reference_sce <- allen_reference_sce[, unlist(subset_cells)]
# check again number of cells per subclass
table(allen_reference_sce$subclass)##
## Astro CR Endo L2_3_IT L4 L5_IT L5_PT
## 20 7 20 20 20 20 20
## L6_CT L6_IT L6b Lamp5 Macrophage Meis2 NP
## 20 20 20 20 20 20 20
## Oligo Peri Pvalb Serpinf1 SMC Sncg Sst
## 20 20 20 20 20 20 20
## Vip VLMC
## 20 20
Then run normalization and dimensionality reduction.
allen_reference_sce <- computeSumFactors(allen_reference_sce, sizes = c(20, 40, 60,
80))
allen_reference_sce <- logNormCounts(allen_reference_sce)
allen.var.out <- modelGeneVar(allen_reference_sce, method = "loess")
allen.hvgs = getTopHVGs(allen.var.out, n = 2000)Since the scRNAseq dataset was generated from the mouse cortex, we will subset the visium dataset in order to select mainly the spots part of the cortex. Note that the integration can also be performed on the whole brain slice, but it would give rise to false positive cell type assignments and and therefore it should be interpreted with more care.
Here, will use SingleR for prediciting which cell types are present in the dataset.
We can first select the anterior part as an example (to speed up predictions).
sce.anterior <- sce[, sce$Sample == "Anterior"]
sce.anterior@metadata <- sce.anterior@metadata[["Anterior"]]Next, we select the highly variable genes that are present in both datasets.
# Find common highly variable genes
common_hvgs <- allen.hvgs[allen.hvgs %in% hvgs]
# Predict cell classes
pred.grun <- SingleR(test = sce.anterior[common_hvgs, ], de.n = 20, ref = allen_reference_sce[common_hvgs,
], labels = allen_reference_sce$subclass, de.method = "wilcox")
# Transfer the classes to the SCE object
sce.anterior$cell_prediction <- pred.grun$labels
sce.anterior@colData <- cbind(sce.anterior@colData, as.data.frame.matrix(table(list(1:ncol(sce.anterior),
sce.anterior$cell_prediction))))Then we can plot the predicted cell populations back to tissue.
# Plot cell predictions
spanielPlot(object = sce.anterior, plotType = "Cluster", clusterRes = "cell_prediction",
customTitle = "cell_prediction", techType = "Visium", ptSizeMax = 1, ptSizeMin = 0.1)plist <- list()
n = 1
for (i in c("L2_3_IT", "L4", "L5_IT", "L6_IT")) {
plist[[n]] <- spanielPlot(object = sce.anterior, plotType = "Cluster", clusterRes = i,
customTitle = i, techType = "Visium", ptSize = 0.3, ptSizeMax = 1, ptSizeMin = 0.1)
n <- n + 1
}
plot_grid(ncol = 2, plotlist = plist)Keep in mind, that the scores are “just” prediction scores, and do not correspond to proportion of cells that are of a certain celltype or similar. It mainly tells you that gene expression in a certain spot is hihgly similar/dissimilar to gene expression of a celltype.
If we look at the scores, we see that some spots got really clear predictions by celltype, while others did not have high scores for any of the celltypes.
We can also plot the gene expression and add filters together, too:
spanielPlot(object = sce.anterior, plotType = "Gene", gene = "Wfs1", showFilter = sce.anterior$L4,
customTitle = "", techType = "Visium", ptSize = 0, ptSizeMin = -0.3, ptSizeMax = 1)sessionInfo()## R version 4.0.3 (2020-10-10)
## Platform: x86_64-apple-darwin13.4.0 (64-bit)
## Running under: macOS Catalina 10.15.5
##
## Matrix products: default
## BLAS/LAPACK: /Users/paulo.czarnewski/.conda/envs/scRNAseq2021/lib/libopenblasp-r0.3.12.dylib
##
## locale:
## [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
##
## attached base packages:
## [1] parallel stats4 stats graphics grDevices utils datasets
## [8] methods base
##
## other attached packages:
## [1] cowplot_1.1.1 patchwork_1.1.1
## [3] scater_1.18.0 ggplot2_3.3.3
## [5] SingleR_1.4.0 scran_1.18.0
## [7] SingleCellExperiment_1.12.0 SummarizedExperiment_1.20.0
## [9] Biobase_2.50.0 GenomicRanges_1.42.0
## [11] GenomeInfoDb_1.26.0 IRanges_2.24.0
## [13] S4Vectors_0.28.0 BiocGenerics_0.36.0
## [15] MatrixGenerics_1.2.0 matrixStats_0.57.0
## [17] dplyr_1.0.3 Matrix_1.3-2
## [19] biomaRt_2.46.0 Spaniel_1.2.0
## [21] RJSONIO_1.3-1.4 optparse_1.6.6
##
## loaded via a namespace (and not attached):
## [1] R.utils_2.10.1 reticulate_1.18
## [3] tidyselect_1.1.0 RSQLite_2.2.2
## [5] AnnotationDbi_1.52.0 htmlwidgets_1.5.3
## [7] BiocParallel_1.24.0 grid_4.0.3
## [9] Rtsne_0.15 DropletUtils_1.10.2
## [11] devtools_2.3.2 munsell_0.5.0
## [13] codetools_0.2-18 ica_1.0-2
## [15] statmod_1.4.35 future_1.21.0
## [17] miniUI_0.1.1.1 batchelor_1.6.0
## [19] withr_2.4.0 colorspace_2.0-0
## [21] knitr_1.30 Seurat_3.2.3
## [23] ROCR_1.0-11 tensor_1.5
## [25] listenv_0.8.0 labeling_0.4.2
## [27] GenomeInfoDbData_1.2.4 polyclip_1.10-0
## [29] farver_2.0.3 bit64_4.0.5
## [31] rhdf5_2.34.0 rprojroot_2.0.2
## [33] parallelly_1.23.0 vctrs_0.3.6
## [35] generics_0.1.0 xfun_0.20
## [37] BiocFileCache_1.14.0 R6_2.5.0
## [39] ggbeeswarm_0.6.0 rsvd_1.0.3
## [41] locfit_1.5-9.4 rhdf5filters_1.2.0
## [43] bitops_1.0-6 spatstat.utils_1.20-2
## [45] DelayedArray_0.16.0 assertthat_0.2.1
## [47] promises_1.1.1 scales_1.1.1
## [49] beeswarm_0.2.3 gtable_0.3.0
## [51] beachmat_2.6.0 globals_0.14.0
## [53] processx_3.4.5 goftest_1.2-2
## [55] rlang_0.4.10 splines_4.0.3
## [57] lazyeval_0.2.2 yaml_2.2.1
## [59] reshape2_1.4.4 abind_1.4-5
## [61] httpuv_1.5.5 tools_4.0.3
## [63] usethis_1.6.3 ellipsis_0.3.1
## [65] RColorBrewer_1.1-2 sessioninfo_1.1.1
## [67] ggridges_0.5.3 Rcpp_1.0.6
## [69] plyr_1.8.6 sparseMatrixStats_1.2.0
## [71] progress_1.2.2 zlibbioc_1.36.0
## [73] purrr_0.3.4 RCurl_1.98-1.2
## [75] ps_1.5.0 prettyunits_1.1.1
## [77] rpart_4.1-15 openssl_1.4.3
## [79] deldir_0.2-9 viridis_0.5.1
## [81] pbapply_1.4-3 zoo_1.8-8
## [83] ggrepel_0.9.1 cluster_2.1.0
## [85] fs_1.5.0 magrittr_2.0.1
## [87] RSpectra_0.16-0 data.table_1.13.6
## [89] scattermore_0.7 ResidualMatrix_1.0.0
## [91] lmtest_0.9-38 RANN_2.6.1
## [93] fitdistrplus_1.1-3 pkgload_1.1.0
## [95] hms_1.0.0 mime_0.9
## [97] evaluate_0.14 xtable_1.8-4
## [99] XML_3.99-0.5 gridExtra_2.3
## [101] testthat_3.0.1 compiler_4.0.3
## [103] tibble_3.0.5 KernSmooth_2.23-18
## [105] crayon_1.3.4 R.oo_1.24.0
## [107] htmltools_0.5.1 mgcv_1.8-33
## [109] later_1.1.0.1 tidyr_1.1.2
## [111] DBI_1.1.1 formatR_1.7
## [113] dbplyr_2.0.0 MASS_7.3-53
## [115] rappdirs_0.3.1 getopt_1.20.3
## [117] cli_2.2.0 R.methodsS3_1.8.1
## [119] igraph_1.2.6 pkgconfig_2.0.3
## [121] scuttle_1.0.0 plotly_4.9.3
## [123] xml2_1.3.2 vipor_0.4.5
## [125] dqrng_0.2.1 XVector_0.30.0
## [127] stringr_1.4.0 callr_3.5.1
## [129] digest_0.6.27 sctransform_0.3.2
## [131] RcppAnnoy_0.0.18 spatstat.data_1.7-0
## [133] rmarkdown_2.6 leiden_0.3.6
## [135] edgeR_3.32.0 uwot_0.1.10
## [137] DelayedMatrixStats_1.12.0 curl_4.3
## [139] shiny_1.5.0 lifecycle_0.2.0
## [141] nlme_3.1-151 jsonlite_1.7.2
## [143] Rhdf5lib_1.12.0 BiocNeighbors_1.8.0
## [145] limma_3.46.0 desc_1.2.0
## [147] viridisLite_0.3.0 askpass_1.1
## [149] fansi_0.4.2 pillar_1.4.7
## [151] lattice_0.20-41 fastmap_1.0.1
## [153] httr_1.4.2 pkgbuild_1.2.0
## [155] survival_3.2-7 glue_1.4.2
## [157] remotes_2.2.0 spatstat_1.64-1
## [159] png_0.1-7 bluster_1.0.0
## [161] bit_4.0.4 HDF5Array_1.18.0
## [163] stringi_1.5.3 blob_1.2.1
## [165] BiocSingular_1.6.0 memoise_1.1.0
## [167] irlba_2.3.3 future.apply_1.7.0